HIV-1 protein Nef is a multifunctional protein involved in regulation of viral infectivity and responsible for many pathogenic effects of HIV infection, including downregulation of CD4, MHC I and ABCA1. Nef-mediated impairment of ABCA1 activity is a key event in pathogenesis of HIV-related atherosclerosis, a leading co-morbidity of HIV disease. While studying Nef-mediated inactivation of the cholesterol transporter ABCA1, we found that this Nef activity involved disruption by Nef of an interaction between ABCA1 and calnexin, an endoplasmic reticulum chaperone regulating folding and maturation of glycosylated proteins. Calnexin is an integral ER transmembrane protein, and preliminary analysis demonstrated that the cytoplasmic domain of calnexin was necessary for its interaction with Nef. We also demonstrated that calnexin-Nef interaction is direct and is thus amenable for crystallization studies. Intriguingly, while interaction between calnexin and ABCA1 was disrupted by Nef, the interaction between calnexin and gp160 was actually enhanced. No effect of Nef on abundance or localization of calnexin was detected, suggesting that Nef regulates interactions of calnexin with its targets in the ER lumen through structural modifications. This proposal is designed to characterize the structural basis of the interaction between Nef and calnexin. The following Specific Aims will be pursued: 1) Preparation and initial pre-crystallographic characterization of the Nef/calnexin complexes; 2) Crystallographic analysis of the Nef/calnexin complexes. Significance of this proposal stems from a demonstrated contribution of the Nef-calnexin interaction to disruption of cellular cholesterol metabolism (through impairment of ABCA1). Therefore, knowledge gained from this study will be essential to our efforts to curb HIV-related cardio-vascular disease.